Method for detecting staphylococcus aureus by using electrochemical luminescence aptamer sensor

Abstract

The invention discloses a method for detecting staphylococcus aureus by using an electrochemical luminescence aptamer sensor. The method comprises the following steps: step 1, synthesizing Au NPs; 2, synthesizing a Ru (bpy) 32 + (at) MXene composite material; step 3, sensor construction and ECL detection: step 3a, polishing the surface of the GCE with Al2O3 polishing powder, washing with ultrapure water, and blow-drying with nitrogen; step 3b, obtaining Ru (bpy) 32 + (at) MXene / GCE; step 3c, obtaining an Au NPs-Ru (bpy) 32 + (at) Mxene membrane electrode; step 3d, dispensing cDNA activated by TCEP on the surface of Au NPs-Ru (bpy) 32 + (at) Mxene / GCE, incubating, and fixing the cDNA on the surface of the electrode through self-assembly of a gold-sulfydryl bond; step 3e, mixing the aptamer with P-DNA / Fc modified with ferrocene (Fc), and carrying out a reaction in a dark place to obtain double-stranded DNA; step 3f, mixing staphylococcus aureus with different solubilities with the double-stranded DNA, soaking the cDNA-modified electrode in the mixed solution, and reacting in a dark place; and immersing the electrode into a 1 * PBS buffer solution containing TPA. The detection sensitivity of pathogenic bacteria can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of Staphylococcus aureus detection. Background Art

[0002] Electrochemiluminescence (ECL) is an effective analytical method that combines the advantages of electrochemistry and chemiluminescence. It has the characteristics of high sensitivity, low background and electrochemical controllability. It has been widely used in the field of clinical detection. Among the many ECL emitters, Ru(bpy)3 2+ and its derivatives have become extremely important ECL luminophores due to their excellent stability and strong ECL signal; however, Ru(bpy)3 2+ It is easy to overflow in the liquid phase, causing a lot of loss. Therefore, during the ECL detection process, Ru(bpy)3 2+ It is necessary and challenging to fix it on the electrode surface to reduce its dissipation in the solution. Nanomaterials can be used as luminescent molecules Ru(bpy)3 due to their many advantages. 2+ Such nanomaterials include but are not limited to silica nanoparticles (SiO2 NPs), carbon nanotubes (CNTs), graphene oxide (GO) and metal organic frameworks (MOFs). 2+ Antibodies were labeled on the surface of doped SiO2 NPs to construct a sandwich-type electrochemiluminescence (ECL) immunosensor. SiO2 NPs were loaded with Ru(bpy)3 2+ At the same time, its porous structure and large specific surface area can fix more antibodies, improving the sensitivity of detection. However, these materials themselves have poor conductivity, which will affect the Ru(bpy)3 on the material to a certain extent. 2+ activation, so a material that can load Ru(bpy)3 2+ MXene nanosheets not only have the advantages of superior conductivity, excellent stability and high surface area, but also have the advantages of -OH, O or -F functional groups on the surface, which can make them negatively charged, so MXene can achieve electrophilic molecule fixation through electrostatic interaction. Utilizing the different properties of MXene and other two-dimensional nanomaterials, MXene is expected to achieve Ru(bpy)3 2+ Stable fixation of derivatives.

[0003] Aptamers have the advantages of easy synthesis and modification, low cost, high affinity and high specificity, and are often used for specific detection of biomarkers. Aptamer-based electrochemical sensors are often used to detect pathogenic bacteria. The sensing process is carried out on the electrode surface, but due to the influence of steric hindrance on the activity of biomolecules, the binding efficiency of biomolecules and the biological recognition efficiency of targets are reduced, making the detection sensitivity of pathogenic bacteria still challenging. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the defects of the prior art and propose a method for detecting Staphylococcus aureus by electrochemiluminescent aptamer sensor, which utilizes the superior conductivity, excellent stability and high surface area of ​​two-dimensional MXene and its abundant negatively charged surface active sites to convert Ru(dcbpy)3 2+ Electrochemical luminescent materials were obtained by adsorption on the surface of MXene materials. A two-phase substitution strategy was adopted to specifically recognize the target through the aptamer and fix the luminescent molecule Ru(dcbpy)3 through MXene. 2+ The electrochemiluminescent aptamer sensor of the present invention is prepared in this way to improve the detection sensitivity of pathogenic bacteria.

[0005] In order to solve the above technical problems, the present invention proposes the following technical solutions: A method for detecting Staphylococcus aureus using an electrochemiluminescent aptamer sensor comprises the following steps:

[0006] Step 1: Synthesis of Au NPs;

[0007] Step 2: Synthesis of Ru(bpy)3 2+ @MXene composites;

[0008] Step 3: Sensor construction and ECL detection, including the following steps:

[0009] Step 3a: The GCE surface was polished with Al2O3 polishing powder and rinsed with ultrapure water, and dried with nitrogen for further use;

[0010] Step 3b: Take Ru(bpy)3 2+ @MXene was dissolved in PEI solution, which was drop-coated on the treated GCE surface and dried to obtain Ru(bpy)3 2+ @MXene / GCE;

[0011] Step 3c: Then, Au NPs were drop-coated on the surface of the Ru(bpy)3 2+ @MXene membrane electrode surface, continue drying to obtain Au NPs-Ru(bpy)3 2+ @Mxene membrane electrode;

[0012] Step 3d: Apply TCEP-activated cDNA droplets onto Au NPs-Ru(bpy)3 2+ @Mxene / GCE surface and incubated, cDNA was immobilized on the electrode surface through gold-thiol self-assembly;

[0013] Step 3e: Mix the aptamer with the P-DNA / Fc modified with ferrocene, and react in the dark to obtain a double-stranded DNA, namely, the aptamer-P-DNA / Fc, which is then stored in the dark for use in the next step;

[0014] Step 3f: Mix different solubilities of Staphylococcus aureus with double-stranded DNA, namely aptamer-P-DNA / Fc, and then immerse the electrode modified with cDNA in the mixed solution to protect the reaction from light; after each modification step, wash the electrode with ultrapure water and dry it with nitrogen; immerse the electrode in 1×PBS buffer containing TPA.

[0015] The above technical solution is further limited in that step 1 of synthesizing Au NPs includes: soaking all glassware and magnets in aqua regia, and then rinsing them with tap water and ultrapure water in turn, heating HAuCl4 under stirring and maintaining for 30 minutes, then quickly adding sodium citrate, and then maintaining the mixture at this temperature and stirring for 20 minutes; then stopping heating, maintaining the condensation reflux state, and gradually cooling to room temperature to obtain a seed solution, which is taken out and stored for later use.

[0016] The above technical solution is further limited in that step 1 of synthesizing Au NPs includes: soaking all glassware and magnets in aqua regia, and then rinsing them with tap water and ultrapure water in turn, heating 100 mL of HAuCl4 (w / w, 0.01%) to 120°C under stirring and maintaining for 30 minutes, then quickly adding 10 mL of sodium citrate (w / w, 1%), and then maintaining the mixture at this temperature and stirring for 20 minutes; then stopping heating, maintaining the condensation reflux state, and gradually cooling to room temperature to obtain a seed solution with a particle size of about 15 nm, which is taken out and stored for later use.

[0017] The above technical solution is further limited in that step 2 synthesizes Ru(bpy)3 2+ @MXene composite material includes: dissolving Ru(dcbpy)3Cl2 in deionized water, adding MXene aqueous solution, mixing with ultrasound, and preparing Ru(bpy)3 by electrostatic adsorption. 2+ @MXene composite materials, then washed with deionized water and redispersed in deionized water and stored in a refrigerator at 4 °C for further use.

[0018] The above technical solution is further limited in that step 2 synthesizes Ru(bpy)3 2+@MXene composite material includes: 9 mg of Ru(dcbpy)3Cl2 was dissolved in 1 mL of deionized water, and then 4 mL of MXene (5 mg / mL) aqueous solution was added, and mixed with ultrasound for 30 s to prepare Ru(bpy)3 by electrostatic adsorption. 2+ @MXene composite material, then washed three times with deionized water and redispersed in deionized water and stored in a refrigerator at 4 °C for further use.

[0019] The above technical solution is further limited in that step 3 of sensor construction and ECL detection comprises the following steps:

[0020] Step 3a: The GCE surface was polished with Al2O3 polishing powder (0.3 μm and 0.05 μm) and rinsed with ultrapure water and dried with nitrogen for further use;

[0021] Step 3b: Take 10 μL of Ru(bpy)3 2+ @MXene (0.5 mg / mL) was dissolved in 200 μL PEI (w / w, 2%) solution, which was drop-coated on the treated GCE surface and dried at 37 °C for 2 h to obtain Ru(bpy)3 2+ @MXene / GCE;

[0022] Step 3c: 4 μL of Au NPs were then dropped onto the Ru(bpy)3 2+ @MXene membrane electrode surface, continue to dry in 37℃ environment for 1h to obtain Au NPs-Ru(bpy)3 2+ @Mxene membrane electrode;

[0023] Step 3d: 10 μL cDNA (1.4 μM) activated with TCEP was drop-coated on Au NPs-Ru(bpy)3 2+ @Mxene / GCE surface and incubated at 37°C for 2 h, cDNA was immobilized on the electrode surface through gold-thiol self-assembly;

[0024] Step 3e: 10 μL of aptamer (2.4 μM) was mixed with 10 μL of P-DNA / Fc modified with ferrocene (2.4 μΜ), and the mixture was reacted at 37°C in the dark for 1 h to obtain double-stranded DNA, namely aptamer-P-DNA / Fc, and stored at 4°C in the dark for the next step;

[0025] Step 3f: Mix 20 μL of Staphylococcus aureus of different solubility with 20 μL of double-stranded DNA, namely aptamer-P-DNA / Fc (1.2 μM), and then immerse the electrode modified with cDNA in the mixed solution and react at 37°C in the dark for 1 hour; after each modification step, wash the electrode with ultrapure water and dry it with nitrogen; immerse the electrode in 1 mL 1×PBS buffer containing 6 μL TPA (10 mM).

[0026] The above technical solution is further limited in that, in step 3f, the potential during ECL detection is in the range of 0.4-1.6V and the scanning rate is 100mV / s.

[0027] The present invention has the following beneficial effects: The present invention prepares Ru(dcbpy)3 by electrostatic adsorption 2+ @MXene luminescent electrode material and combined it with the two-phase substitution strategy to develop a sensitive electrochemiluminescent aptamer sensor to achieve specific detection of Staphylococcus aureus. Unlike the traditional detection method of directly capturing on the electrode surface, the use of two-phase substitution can greatly reduce the steric hindrance of Staphylococcus aureus on the electrode surface. The sensor of the present invention achieves highly sensitive electrochemiluminescent detection of Staphylococcus aureus, with LOD as low as 6CFU / mL and a linear range of 20-2×10 8 CFU / mL; this strategy has good stability and recovery rate for Staphylococcus aureus in serum. The aptamers used in the sensor of the present invention can be replaced with aptamers of other bacteria, providing a new perspective for the future development of detection and rapid, accurate and sensitive analysis of bacterial pathogens.

[0028] In the present invention, the superior conductivity, excellent stability, high surface area and abundant negatively charged surface active sites of two-dimensional MXene are utilized to electrostatically bind Ru(dcbpy)3 2+ Electrochemical luminescent materials were obtained by adsorption on the surface of MXene materials. In order to reduce the influence of steric hindrance on the electrode surface caused by the large volume of the target Staphylococcus aureus, this work adopted a two-phase substitution strategy, using aptamers to specifically recognize the target and MXene to fix the luminescent molecule Ru(dcbpy)3 2+ In this way, a pair of electrochemiluminescent aptamer sensors were developed for highly sensitive and specific detection of Staphylococcus aureus. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The schematic diagram is a schematic diagram of the electrochemiluminescent aptamer sensor of the present invention used for detecting Staphylococcus aureus.

[0030] Figure 2The TEM image of Au NPs (part A in the figure) and the UV-visible spectrum of Au NPs (part B in the figure) are shown.

[0031] Figure 3 MXene and Ru(dcbpy)3 2+ @MXene UV-visible spectrum (part A in the figure), Ru(dcbpy)3 2+ @MXene SEM image and elemental mapping (part B in the figure), Ru(dcbpy)3 2+ EDS spectra of @MXene (part C in the figure) and Au NPs-Ru(dcbpy)3 2+ @SEM image of MXene (part D in the figure).

[0032] Figure 4 It is the polyacrylamide gel electrophoresis diagram (part A in the figure), the EIS diagram of the electrode modification process (part B in the figure) and the ECL signal diagram of the sensor (part C in the figure).

[0033] Figure 5 : is a graph showing the relationship between ΔECL signal and concentration, wherein part A in the graph is the concentration of cDNA, and part B in the graph is the concentration of P-DNA / Fc and aptamer.

[0034] Figure 6 The relationship between ΔECL and the concentration of Staphylococcus aureus (part A in the figure), the linear relationship between ΔECL and the logarithm of the concentration of Staphylococcus aureus (part B in the figure), and the concentration of 10 5 The corresponding signal diagrams of Escherichia coli (Ⅰ), Staphylococcus aureus (Ⅱ), Salmonella typhimurium (Ⅲ) and Pseudomonas aeruginosa (Ⅳ) and buffer solution (Ⅴ) (part C in the figure) and the corresponding signal diagrams of Escherichia coli (Ⅰ), Staphylococcus aureus (Ⅱ), Salmonella typhimurium (Ⅲ) and Pseudomonas aeruginosa (Ⅳ) at 2×10 2 CFU / mL, 2×10 5 CFU / mL, 2×10 6 The ECL signal graph of three parallel measurements at a concentration of 1.5 CFU / mL (part D in the figure). DETAILED DESCRIPTION

[0035] See also Figures 1 to 6 The present invention provides a method for detecting Staphylococcus aureus using an electrochemiluminescent aptamer sensor, comprising the following steps:

[0036] Step 1: Synthesis of Au NPs;

[0037] The synthesis of gold nanoparticles refers to the method previously reported. Before synthesis, all glassware and magnets were first soaked in aqua regia, and then rinsed with tap water and ultrapure water in turn. 100mL HAuCl4 (w / w, 0.01%) was heated to 120°C with stirring and maintained for 30 minutes, and then 10mL sodium citrate (w / w, 1%) was quickly added, and then the mixture was maintained at this temperature and stirred for 20 minutes; then the heating was stopped, the condensation reflux state was maintained, and the temperature was gradually reduced to room temperature to obtain a seed solution with a particle size of about 15nm, which was taken out and stored for later use.

[0038] Step 2: Synthesis of Ru(bpy)3 2+ @MXene composites;

[0039] First, 9 mg of Ru(dcbpy)3Cl2 was dissolved in 1 mL of deionized water, and then 4 mL of MXene (5 mg / mL) aqueous solution was added and mixed with ultrasound for 30 s to prepare Ru(bpy)3 by electrostatic adsorption. 2+ @MXene composite material, then washed three times with deionized water and redispersed in deionized water and stored in a refrigerator at 4 °C for further use.

[0040] Step 3: Sensor construction and ECL detection, including the following steps:

[0041] Step 3a: polishing the surface of the glassy carbon electrode (GCE) with Al2O3 polishing powder (0.3 μm and 0.05 μm) and rinsing it with ultrapure water, and drying it with nitrogen gas for further use;

[0042] Step 3b: Take 10 μL of Ru(bpy)3 2+ @MXene (0.5 mg / mL) was dissolved in 200 μL PEI (polyetherimide) (w / w, 2%) solution, which was drop-coated on the treated GCE surface and dried at 37 °C for 2 h to obtain Ru(bpy)3 2+ @MXene / GCE;

[0043] Step 3c: 4 μL of Au NPs were then dropped onto the Ru(bpy)3 2+ @MXene membrane electrode surface, continue to dry in 37℃ environment for 1h to obtain Au NPs-Ru(bpy)3 2+ @Mxene membrane electrode;

[0044] Step 3d: 10 μL cDNA (1.4 μM) activated with TCEP (Tris(2-carboxyethyl)phosphine) was drop-coated on the Au NPs-Ru(bpy)3 2+ @Mxene / GCE surface and incubated at 37°C for 2 h, cDNA was immobilized on the electrode surface through gold-thiol self-assembly;

[0045] Step 3e: 10 μL of aptamer (2.4 μM) was mixed with 10 μL of P-DNA / Fc (2.4 μΜ) modified with ferrocene (Fc), and the mixture was reacted at 37°C in the dark for 1 h to obtain double-stranded DNA (i.e., aptamer-P-DNA / Fc), which was then stored in the dark at 4°C for use in the next step;

[0046] Step 3f: Mix 20 μL of Staphylococcus aureus of different solubility with 20 μL double-stranded DNA (i.e., aptamer-P-DNA / Fc) (1.2 μM), and then immerse the electrode modified with cDNA in the mixed solution and react at 37°C in the dark for 1 hour; after each modification step, wash the electrode with ultrapure water and dry it with nitrogen; immerse the electrode in 1 mL 1×PBS buffer containing 6 μL TPA (10 mM); during ECL detection, the potential was in the range of 0.4-1.6 V and the scanning rate was 100 mV / s.

[0047] In the above method, Ru(dcbpy)3Cl2 and tripropylamine (TPA) were purchased from Nakai Technology Co., Ltd.; 20× phosphate buffer (PBS) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; citric acid, chloroauric acid, and sodium borohydride were purchased from Sinopharm Chemical Reagent Co., Ltd.; MXene was purchased from Fuzhou Kehong Technology Co., Ltd., 1×NE Buffer 2 (B2) and HPLC-purified nucleic acid sequences were purchased from Shanghai Shenggong Biotechnology Co., Ltd.; Staphylococcus aureus was provided by the School of Biological Engineering, Fuzhou University; all reagents involved in the experiment were of analytical grade, and ultrapure water (Milli-Q, Millipore) with a resistance of 18.2 MΩ / cm was used throughout the experiment.

[0048] The DNA sequences used in this experiment were purchased from Shanghai Sangon Biotechnology Co., Ltd. Experimental DNA sequences:

[0049] cDNA:5'-SH-(CH2)6-CCCCAAGTGCACGCTACTTTGCTAA-3'

[0050] P-DNA:5'-TTAGCAAAGTAGCGTGCACTTTT-Fc-3'

[0051] Aptamer:5'-CTCGGCACGTTCTCAGTAGCGCTCGCTGGTCATCCCACAGC

[0052] TACGTCAAAAGTGCACGCTACTTTGCTAA-3'

[101]

[0053] The principle of the electrochemiluminescent aptamer sensor of the present invention is described as follows:

[0054] The present invention constructs a dual-phase replacement electrochemiluminescent aptamer sensor based on MXene adsorbing luminescent molecule ruthenium. Figure 1 As shown, MXene with superior conductivity, excellent stability, high surface area and abundant negatively charged surface active sites is used as an electrochemiluminescent sensing platform. Luminescent molecule Ru(bpy)3 2+ Ru(bpy)3 was obtained by adsorption on the MXene surface through electrostatic interaction 2+ @MXene composite materials can reduce its high overflow in the solution; Ru(bpy)3 2+ @MXene and Au NPs were drop-coated on the electrode surface, and then cDNA was modified on the electrode surface through gold-thiol bonds. In order to reduce the influence of steric hindrance on the electrode surface caused by the large size of the target Staphylococcus aureus, the sensor adopted a two-phase substitution strategy: the aptamer in the double-stranded (aptamer-P-DNA / Fc) tends to bind to Staphylococcus aureus to form an aptamer-Staphylococcus aureus complex, resulting in the disintegration of the double-stranded (aptamer-P-DNA / Fc) and the release of P-DNA / Fc; P-DNA / Fc replaces Staphylococcus aureus to approach the electrode surface and bind to the cDNA on the electrode. Since ferrocene (Fc) can inhibit Ru(bpy)3 2+ When P-DNA / Fc is close to the electrode surface, the ECL signal on the electrode surface will be suppressed. Therefore, the detection of Staphylococcus aureus can be achieved through the change of signal.

[0055] The material characterization of the present invention is introduced as follows:

[0056] Au NPs were characterized by UV-visible absorption spectroscopy and TEM images. Figure 2 As shown in part A, AuNPs are spherical particles with relatively uniform size and a diameter of about 20 nm; Figure 2 As shown in part B, the maximum absorption peak of Au NPs is located at 530 nm in the UV-visible spectrum, indicating that Au NPs were successfully synthesized.

[0057] Secondly, the synthesized Ru(bpy)3 2+@MXene is characterized. Figure 3 Part A shows Ru(bpy)3 2+ There is an obvious absorption peak at 465nm, which is Ru(bpy)3 2+ Characteristic absorption peak; Ru(bpy)3 2+ @MXene absorption peak compared to Ru(bpy)3 2+ The characteristic absorption peak of Ru(bpy)3 2+ Successfully adsorbed on MXene. Figure 3 As shown in part B, MXene is in the form of flakes, and the elemental mapping spectrum shows that the MXene nanosheets contain Ru elements and are evenly distributed, indicating that Ru(bpy)3 is adsorbed on the surface of MXene. 2+ ;like Figure 3 As shown in part C, Ru(bpy)3 2+ @MXene composite materials contain Ru, Ti, and C elements. The above spectra all indicate that the ECL bioprobe was successfully prepared. 2+ @MXene composites are characterized. Figure 3 As shown in part D, Au NPs are well dispersed in Ru(bpy)3 2+ @MXene surface, and the size of AuNPs is relatively uniform.

[0058] Feasibility analysis of the method for detecting Staphylococcus aureus using the electrochemiluminescent aptamer sensor of the present invention:

[0059] In order to prove that the experiment of the present invention is feasible, we first use polyacrylamide gel electrophoresis to verify the experiment. Figure 4 As shown in Part A, lanes 1 and 2 are the bands when cDNA and P-DNA / Fc are present, respectively; lane 3 is the DNA band of aptamer-P-DNA / Fc, which shows that P-DNA / Fc and aptamer have successfully hybridized; lane 4 is the band after double-stranded DNA reacts with Staphylococcus aureus and then cDNA is added. Comparing lanes 3 and 4, the double-stranded DNA band in lane 4 disappears and a new band appears, which indicates that P-DNA / Fc is released and combines with cDNA to generate ds(cDNA-P-DNA / Fc). Figure 4 As shown in part B, curve a corresponds to GCE, and curve b corresponds to Au NPs-Ru(dcbpy)3 2+ @MXene / GCE, curve c corresponds to cDNA-Au NPs-Ru(dcbpy)3 2+@MXene / GCE, curve d corresponds to P-DNA / Fc-cDNA-Au NPs-Ru(dcbpy)3 2+ @MXene / GCE. The resistance of the unmodified glassy carbon electrode (curve a) is low; the resistance of the modified AuNPs-Ru(bpy)32+@MXene is significantly reduced (curve b), which is because MXene has good conductivity and promotes electron transfer. Subsequently, when cDNA is modified on the electrode, the resistance of the electrode increases (curve c), which is mainly because the nucleic acid backbone hinders the [Fe(CN)6] 3- / 4- When the target is added to the system and binds to the aptamer in the double-stranded DNA, P-DNA / Fc is released and binds to the cDNA on the electrode surface, resulting in an increase in the nucleic acid skeleton on the electrode surface, further hindering [Fe(CN)6] 3- / 4- When it reaches the electrode surface, the resistance increases (curve d).

[0060] The feasibility of the sensor was verified by electrochemiluminescence. Figure 4 As shown in part C, curve a corresponds to GCE, and curve b corresponds to cDNA-Au NPs-Ru(dcbpy)3 2+ @MXene / GCE; Curve c corresponds to Staphylococcus aureus / P-DNA / Fc-cDNA-Au NPs-Ru(dcbpy)3 2+ @MXene / GCE. Curve a is the bare GCE, where almost no electrochemiluminescence occurs; Curve b is the electrode surface modified with Au NPs-Ru(bpy)3 2+ The ECL signal of the @MXene composite material shows that the ECL signal is significantly enhanced after the modified material is modified; the c curve shows that the ECL signal on the electrode surface is significantly reduced after the target appears, indicating that the P-DNA / Fc of the double-stranded DNA in the solution is released and combined with the cDNA on the electrode surface, causing ferrocene (Fc) to approach the electrode surface and inhibit Ru(bpy)3 2+ The above results show that the proposed experimental scheme is feasible.

[0061] Optimization of the experimental conditions of the present invention:

[0062] In order to obtain the best experimental results of the sensor, several experimental conditions were optimized. Since the concentration of cDNA on the electrode has a direct impact on the ECL signal, when the concentration of cDNA is low, the amount of P-DNA / Fc complementary to it will also decrease, and the obtained ΔECL signal will be low; as the cDNA increases, the ΔECL signal will also increase, so the concentration of cDNA is optimized first. Figure 5As shown in part A, the ΔECL signal in the system increases with the increase of cDNA concentration. When the concentration of cDNA exceeds 1.4 μM, the ΔECL in the system tends to be stable, and the optimal concentration of cDNA is 1.4 μM. Then, the concentration of the double-stranded (aptamer-P-DNA / Fc) in the solution is optimized. Figure 5 As shown in Part B, it can be seen that when the concentration of the double-stranded (aptamer-P-DNA / Fc) is 1.2 μM, ΔECL reaches the maximum value, so the optimal concentration of the double-stranded (aptamer-P-DNA / Fc) is 1.2 μM.

[0063] Performance characterization of the electrochemiluminescent aptamer sensor of the present invention:

[0064] Under the optimal conditions, the analytical performance of the electrochemiluminescence sensor was evaluated at different Staphylococcus aureus concentrations. Figure 6 As shown in part A, the ECL signal gradually decreases with the increase of Staphylococcus aureus concentration. Figure 6 As shown in part B, at 20-2×10 8 Within the concentration range of CFU / mL, ΔECL and the logarithm of the Staphylococcus aureus concentration showed a good linear relationship. The linear regression equation is:

[0065] ΔECL=597.4logC-595.4, R2=0.9931

[0066] Where ΔECL is the signal difference of ECL, C is the concentration of Staphylococcus aureus (CFU / mL), and R is the correlation coefficient. The detection limit of the biosensor is as low as 6 CFU / mL (LOD = 3s / k, k is the slope of the calibration curve, and s is the standard deviation of the blank).

[0067] Selectivity and reproducibility are also important parameters for evaluating the practicality of biosensors. In order to verify the selectivity of the proposed sensor, Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa were selected as interferents at a concentration of 10 5 CFU / mL. The concentration of the target Staphylococcus aureus is 10 5 CFU / mL. Figure 6As shown in the middle C part, I in the figure corresponds to Escherichia coli (105CFU / mL); II in the figure corresponds to Pseudomonas aeruginosa (105CFU / mL); III in the figure corresponds to Salmonella typhimurium (105CFU / mL); IV in the figure corresponds to Staphylococcus aureus; V in the figure corresponds to PBS buffer. The response of the interfering substance is low, and a significant ECL signal change can be detected when Staphylococcus aureus is present. The above results show that the electrochemical sensor has good selectivity for Staphylococcus aureus, which is mainly due to the specific interaction between Staphylococcus aureus and the aptamer. Secondly, the reproducibility of the ECL sensor was investigated.

[0068] like Figure 6 As shown in the D part, at 2×10 2 CFU / mL, 2×10 5 CFU / mL, 2×10 6 The concentration of CFU / mL was measured three times, and the RSD of each concentration group was 2.1%, 1.7% and 2.9%, respectively, indicating that the sensor has good reproducibility.

[0069] In order to evaluate the application of the designed electrochemiluminescence sensor in actual sample analysis, the method was used to detect Staphylococcus aureus in normal human serum samples. First, the serum sample was diluted 10 times with phosphate buffered saline (pH = 7.4, 0.1M), and different concentrations of Staphylococcus aureus were added to the serum sample. As shown in Table 1, the recovery rate of Staphylococcus aureus detected by the electrochemiluminescence method was 92.2-108.1%, and the relative standard deviation was less than 8.6%. The results show that the electrochemiluminescence method of the present invention can realize the detection of Staphylococcus aureus in normal human serum samples.

[0070] Table 1 Determination results of Staphylococcus aureus content in normal human serum (n=3)

[0071]

[0072] The present invention has the following beneficial effects: The present invention prepares Ru(dcbpy)3 by electrostatic adsorption 2+ @MXene luminescent electrode material and combined it with the two-phase substitution strategy to develop a sensitive electrochemiluminescent aptamer sensor to achieve specific detection of Staphylococcus aureus. Unlike the traditional detection method of directly capturing on the electrode surface, the use of two-phase substitution can greatly reduce the steric hindrance of Staphylococcus aureus on the electrode surface. The sensor of the present invention achieves highly sensitive electrochemiluminescent detection of Staphylococcus aureus, with LOD as low as 6CFU / mL and a linear range of 20-2×10 8CFU / mL; this strategy has good stability and recovery rate for Staphylococcus aureus in serum. The aptamers used in the sensor of the present invention can be replaced with aptamers of other bacteria, providing a new perspective for the future development of detection and rapid, accurate and sensitive analysis of bacterial pathogens.

[0073] In the present invention, the superior conductivity, excellent stability, high surface area and abundant negatively charged surface active sites of two-dimensional MXene are utilized to electrostatically bind Ru(dcbpy)3 2+ Electrochemical luminescent materials were obtained by adsorption on the surface of MXene materials. In order to reduce the influence of steric hindrance on the electrode surface caused by the large volume of the target Staphylococcus aureus, this work adopted a two-phase substitution strategy, using aptamers to specifically recognize the target and MXene to fix the luminescent molecule Ru(dcbpy)3 2+ In this way, a pair of electrochemiluminescent aptamer sensors were developed for highly sensitive and specific detection of Staphylococcus aureus.

Claims

1. A method for detecting Staphylococcus aureus using an electrochemiluminescent aptamer sensor, characterized in that: It includes the following steps: Step 1: Synthesis of Au NPs; Step 2: Synthesis of Ru(bpy)3 2+ @MXene composites; Step 3: Sensor construction and ECL detection, including the following steps: Step 3a: The GCE surface was polished with Al2O3 polishing powder and rinsed with ultrapure water, and dried with nitrogen for further use; Step 3b: Take Ru(bpy)3 2+ @MXene was dissolved in PEI solution, which was drop-coated on the treated GCE surface and dried to obtain Ru(bpy)3 2+ @MXene / GCE; Step 3c: Then, Au NPs were drop-coated on the surface of the Ru(bpy)3 2+ @MXene membrane electrode surface, continue drying to obtain AuNPs-Ru(bpy)3 2+ @Mxene membrane electrode; Step 3d: Apply TCEP-activated cDNA droplets onto Au NPs-Ru(bpy)3 2+ @Mxene / GCE surface and incubated, cDNA was immobilized on the electrode surface through gold-thiol self-assembly; Step 3e: Mix the aptamer with the P-DNA / Fc modified with ferrocene, and react in the dark to obtain a double-stranded DNA, namely, the aptamer-P-DNA / Fc, which is then stored in the dark for use in the next step; Step 3f: Mix different solubilities of Staphylococcus aureus with double-stranded DNA, namely aptamer-P-DNA / Fc, and then immerse the electrode modified with cDNA in the mixed solution to protect the reaction from light; after each modification step, wash the electrode with ultrapure water and dry it with nitrogen; immerse the electrode in 1×PBS buffer containing TPA.

2. The method for detecting Staphylococcus aureus using an electrochemiluminescent aptamer sensor according to claim 1, characterized in that: Step 1: Synthesizing Au NPs includes: soaking all glassware and magnets with aqua regia, then rinsing with tap water and ultrapure water in turn, heating HAuCl4 with stirring and maintaining for 30 minutes, then quickly adding sodium citrate, and then maintaining the mixture at this temperature and stirring for 20 minutes; then stopping heating, maintaining the condensation reflux state, and gradually cooling to room temperature to obtain a seed solution, which is taken out and stored for later use.

3. The method for detecting Staphylococcus aureus using an electrochemiluminescent aptamer sensor according to claim 1 or 2, characterized in that: Step 1: Synthesizing Au NPs includes: soaking all glassware and magnets with aqua regia, and then rinsing with tap water and ultrapure water in turn, heating 100 mL of HAuCl4 (w / w, 0.01%) to 120°C under stirring and maintaining for 30 min, then quickly adding 10 mL of sodium citrate (w / w, 1%), and then maintaining the mixture at this temperature and stirring for 20 min; then stopping heating, maintaining the condensation reflux state, and gradually cooling to room temperature to obtain a seed solution with a particle size of about 15 nm, which is taken out and stored for later use.

4. The method for detecting Staphylococcus aureus using an electrochemiluminescent aptamer sensor according to claim 1, characterized in that: Step 2 Synthesis of Ru(bpy)3 2+ @MXene composite material includes: dissolving Ru(dcbpy)3Cl2 in deionized water, adding MXene aqueous solution, mixing with ultrasound, and preparing Ru(bpy)3 by electrostatic adsorption. 2+ @MXene composite materials, then washed with deionized water and redispersed in deionized water and stored in a refrigerator at 4 °C for further use.

5. The method for detecting Staphylococcus aureus using an electrochemiluminescent aptamer sensor according to claim 1 or 4, characterized in that: Step 2 Synthesis of Ru(bpy)3 2+ @MXene composite material includes: 9 mg of Ru(dcbpy)3Cl2 was dissolved in 1 mL of deionized water, and then 4 mL of MXene (5 mg / mL) aqueous solution was added, and mixed with ultrasound for 30 s to prepare Ru(bpy)3 by electrostatic adsorption. 2+ @MXene composite material, then washed three times with deionized water and redispersed in deionized water and stored in a refrigerator at 4 °C for further use.

6. The method for detecting Staphylococcus aureus using an electrochemiluminescent aptamer sensor according to claim 1, characterized in that: Step 3: Sensor construction and ECL detection, including the following steps: Step 3a: The GCE surface was polished with Al2O3 polishing powder (0.3 μm and 0.05 μm) and rinsed with ultrapure water and dried with nitrogen for further use; Step 3b: Take 10 μL of Ru(bpy)3 2+ @MXene (0.5 mg / mL) was dissolved in 200 μL PEI (w / w, 2%) solution, which was drop-coated on the treated GCE surface and dried at 37 °C for 2 h to obtain Ru(bpy)3 2+ @MXene / GCE; Step 3c: 4 μL of Au NPs were then dropped onto the Ru(bpy)3 2+ @MXene membrane electrode surface, continue to dry in 37℃ environment for 1h to obtain Au NPs-Ru(bpy)3 2+ @Mxene membrane electrode; Step 3d: 10 μL cDNA (1.4 μM) activated with TCEP was drop-coated on Au NPs-Ru(bpy)3 2+ @Mxene / GCE surface and incubated at 37°C for 2 h, cDNA was immobilized on the electrode surface through gold-thiol self-assembly; Step 3e: 10 μL of aptamer (2.4 μM) was mixed with 10 μL of P-DNA / Fc modified with ferrocene (2.4 μΜ), and the mixture was reacted at 37°C in the dark for 1 h to obtain double-stranded DNA, namely aptamer-P-DNA / Fc, and stored at 4°C in the dark for the next step; Step 3f: Mix 20 μL of Staphylococcus aureus of different solubility with 20 μL of double-stranded DNA, namely aptamer-P-DNA / Fc (1.2 μM), and then immerse the electrode modified with cDNA in the mixed solution and react at 37°C in the dark for 1 hour; after each modification step, wash the electrode with ultrapure water and dry it with nitrogen; immerse the electrode in 1 mL 1×PBS buffer containing 6 μL TPA (10 mM).

7. The method for detecting Staphylococcus aureus using an electrochemiluminescent aptamer sensor according to claim 1 or 6, characterized in that: In step 3f, the potential during ECL detection was in the range of 0.4-1.6 V, and the scan rate was 100 mV / s.

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